Study On The Fermentation And Preparation Of Siderophores From Synechococcus Sp. PCC 7002

In order to improve the yield of siderophores from Synechococcus sp. PCC 7002 and get a set of technology for siderophores preparation, detection and separation methods of siderophores was investigated in this paper. Single factor experiment and response surface methodology(RSM) were used to optimize the fermentation conditions to improve the yield of the siderophores from Synechococcus sp. PCC 7002. Amberlite XAD-2 resin and immobilized metal-chelating affinity chromatography(IMAC) were used for siderophores separation. The fermentation and preparation of siderophores made it possible for its widespread use.This paper included the following three parts:1. Detection and identification of the siderophores from Synechococcus sp. PCC 7002:Two-layer plate, mixture plate and traditional chromazurol S plate were used to detect the siderophores from Synechococcus sp. PCC 7002 qualitatively. The results showed that the mixture plate method was more rapid, efficient and convenient among the three qualitative detection methods. It was the optimal method for siderophores qualitative detection. The relative amount of siderophores was detected by chromazurol S shuttle assay. Curve of the relationship between the relative amount and molar mass of the siderophores was drawn to provide theoretical basis for quantitative detection of siderophores. Arnow test, Shenker test and Csaky test were used to identify the siderophores from Synechococcus sp. PCC 7002, the results indicated that it is a hydroxamate-type siderophore. Standard curve of hydroxylamine hydrochloride method was drawn for Csaky test.2. Optimization of the fermentation conditions for siderophores from Synechococcus sp. PCC 7002In this paper, single factor experiment was used to optimize the fermentation conditions included iron concentration, pH, temperature, carbon sources, nitrogen sources, bottle filling amount and phosphorus content. The results showed that the yield of siderophores was higher and higher with the bottling amount decreasing. It has the highest yield when the medium without any iron content. The optimum culture conditions for siderophores fermentation were as follows:pH 8.0, temperature 30℃, the optimal carbon source was 15 g/L glucose, the optimum nitrogen source was 0.2 g/L NaNO3 and 0.15 g/L NH4Cl. The effect of phosphate concentration on the siderophores content was not significant, so the phosphate concentration of the medium was not adjusted.On the basis of single factor experiment, Box-Behnken RSM was employed with four factors such as temperature. pH, carbon sources, nitrogen sources on the yield of siderophores. The optimum culture conditions were as follows:the temperature was 31.7℃, pH was 8.28, the amount of glucose was 14.47 g/L, the amount of NH4Cl was 0.19 g/L. The effect of temperature, pH and glucose on siderophores content was significant and the effect of NH4Cl was not significant. Confirmatory experiment was used to test the results provided by RSM. The relative amount of siderophores from Synechococcus sp. PCC 7002 was 95.42% under the optimum culture conditions after 15 days fermentation while the other group without optimizing was only 64.53%. The relative amount of siderophores increased 30.89% after optimizing. The relative error between measured value and theoretical value was 4.58%< 5%. The culture conditions obtained by RSM were effective and reliable, the yield of siderophores improved significantly.3. Separation of the siderophores from Synechococcus sp. PCC 7002In this paper, Amberlite XAD-2 resin and IMAC were used for siderophores separation. It was simple, convenient and economic for the macroporous resin method because of the reused filler. Siderophores adsorbed sufficiently into Amberlite XAD-2 resin and the separating efficiency was relatively high when the sample pH was adjusted to 2.Two different elution methods (pH reduced elution and competitive elution) utilized in IMAC can both separate the siderophores from Synechococcus sp. PCC 7002 significantly. PBS was used as the elution buffer in IMAC separation. It would destroy the stability of chromazurol S shuttle assay when the concentration of PBS was very high. So the siderophores separated by IMAC was detected directly without rotary evaporation. The separation effect of IMAC was very good. This method can be used to separate pure siderophores for structure research.